Galactosemia is a life threatening inborn error of metabolism. It is caused by a deficiency in the enzyme galactose-1-phosphate uridyl transferase (GALT). GALT is important in the metabolism of galactose and it catalyses an important step in the conversion of galactose-1-phosphate (GAL-1-P) to glucose-6-phosphate (GLC-6-P). In patients with galactosemia the conversion is very low or non-existing leading to harmful levels of GAL-1-P and metabolites thereof.
In many countries galactosemia screening is part of the national newborn screening programmes. At present, an enzyme activity test (i.e. Beutler test) is the predominant method used for galactosemia screening (Beutler E, Baluda M C. A simple spot screening test for galactosemia. J Lab Clin Med 1966; 68:137-141). The main advantage to the Beutler test is that it measures enzyme activity rather than metabolite concentrations. The main disadvantage is that it requires the enzyme activity to be intact. Enzymes are sensitive to heat and can be rendered inactive by excessive heat thus leading to false positives. Also, the Beutler test calls for a sample of its own. It is not possible to multiplex it with other screening tests.
It is also possible use metabolites as biomarkers when screening for galactosemia. Biomarker based screening for galactosemia using mass spectrometry has previously been described in WO0210740. Briefly, elevated levels of hexose-monophosates are used as biomarkers for the disease. This is based on the fact that galactosemia leads to elevated levels of GAL-1-P, which is a hexose-monophosphate. Ideally, screening should be based on GAL-1-P alone. However, in mass spectrometry (MS) it is not possible to differentiate between different types of hexose-monophosphates. It is possible to use liquid chromatography prior to mass spectral determination (LC-MS) in order to differentiate between them but this is not practicable in newborn screening due to the demands of high sample throughput. The rational for using total hexose-monophosphate as a biomarker is that in patients with galactosemia GAL-1-P levels becomes elevated above the levels of all other hexose-monophosphates. An abnormal concentration of total hexose-monophosphates is definitely indicative of galactosemia. This approach is effective in detecting patients who are receiving galactose containing food. Breast milk, cow milk and milk based foods contain lactose which consists of a galactose and a glucose unit joined by a 1,1-glucoside bond. It is quickly converted to galactose and glucose in the digestive tract. Neonates on a normal diet are therefore exposed to high levels of galactose and if they have galactosemia their levels of GAL-1-P will increase dramatically.
However, at birth several types of hexose-monophosphates are present at low levels in neonates, predominantly GLC-6-P. The presence of GLC-6-P causes a background level in the measurement of total hexose-monophosphates which in turn has a detrimental effect on the specificity and sensitivity of the screening method. For the hexose-monophosphate method to function properly the levels of GAL-1-P have to be substantially above the levels of GLC-6-P in order to be able to detect a positive sample. Although the levels of GAL-1-P are elevated at birth in patients with galactosemia the levels only rise dramatically after several days of ingesting food containing galactose. Typically a steady state is obtained after 3 to 5 days after birth. The fact that it is necessary for the child to have ingested a significant amount of galactose prior to sampling is a major drawback in screening. In many countries newborn screening samples are taken before 3 to 5 days, also some newborns have feeding problems and reach steady state levels at a later time.
As mentioned earlier, GAL-1-P levels are elevated in galactosemia patients already at birth. If the background created by the presence of GLC-6-P could be removed it would be possible to screen for galactosemia in newborns. The present invention provides a mean to chemically deplete GLC-6-P without affecting GAL-1-P thereby making it possible to screening for galactosemia using mass spectrometric measurement of GAL-1-P independent of sampling time. A further advantage is that newborn screening with MS allows for multiplexing, i.e. it is possible to combine several screening test in one sample.